Clinical Significance of Pregnanediol Excretion Joseph Rogers, M.D.
is the urinary excretion product of the corpus luteum hormone, progesterone. The development of relatively simple methods for the measurement of pregnanediol has led to numerous studies of this steroid in relation to the metabolism of progesterone. It is the purpose of this paper briefly to review current concepts of the significance of pregnanediol excretion.
Marrian in 1929 isolated an inactive steroid alcohol from the urine of pregnant women and determined its molecular formula. This compound was named pregnanediol by Butenandt. Subsequent to the isolation and purification of progesterone in 1934 it was postulated on the basis of the similarity of the chemical structures of the two compounds that pregnanediol might be a metabolic product of progesterone. In 1936 Venning and Browne37 isolated pregnanediol from pregnancy urine as a water-soluble complex salt, sodium pregnanediol glucuronidate, and in 1937 Venning and co-workers41 isolated the compound during the luteal phase of the menstrual cycle and reported recovery of pregnanediol in the urine after intramuscular injection of progesterone. These observations served to establish pregnanediol as the urinary metabolite of progesterone. Subsequently, Vennint4 published a method for the determination From the Pratt Diagnostic Clinic, New England Center Hospital, and Boston Dispensary and the Departments of Internal Medicine and Gynecology, Tufts College Medical School, Boston, Mass. Assistant professor of medicine and lecturer in gynecology, Tufts College Medical School. 513
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of sodium pregnanediol glucuronidate and there developed a large volume of literature on the excretion of pregnanediol, using this method of determination.
MEASUREMENT In 1941 Astwood and Jones introduced a method by which pregnanediol is recovered in the free state rather than as the complex sodium pregnanediol glucuronidate (N aPG ). In the last few years this method or varieties of it for measuring free pregnanediol have proved increasingly popular. 7 • 1o • 14. 220.127.116.11 The method in use in this laboratory is a modification developed by Astwood, Grady, and Wermer of the method reported earlier by Astwood and Jones. Procedure A 24-hour urine is obtained. If the volume is more than 1500 cc. an aliquot is used. The volume of urine is measured and poured into a round-bottom flask, lOO cc. of toluene is added, and then 1 cc. of IBN sulfuric acid for each lO cc. of urine. The flask is attached to a reflux condenser and the contents boiled for 15 minutes. The flame is removed and the flask allowed to cool with gentle shaking until boiling ceases. The ingredients are poured into a separating funnel, shaken, and the toluene extract allowed to separate before discarding the urine. The toluene extract is washed twice with 2N sodium hydroxide and twice with hot distilled water (50 cc. each time). The extract is poured into an Erlenmeyer flask and excess water is boiled off on a hot plate. A column is made using aluminum oxide (mf Gm. water/l00 Gm.) about %inch deep in a 2-cm. diameter sintered glass funnel. Twenty cc. of toluene is added, the aluminum is suspended by vigorous mixing with a spatula and then allowed to settle. The aluminum oxide is covered with a layer of sand, care being taken not to disturb the column. The toluene extract is drawn through the column by gentle suction, and the column is then washed with lO cc. of toluene. Blue and green fractions will come through with the toluene. The column is washed with 0.5% absolute alcohol in toluene until all the pink color is washed through. These fractions are discarded. Three per cent absolute alcohol in toluene is then used to remove the next fraction containing the pregnanediol, the color of which ranges from yellow to brown. The column is washed with approximately 100 cc. alcohol to be sure that all the pregnanediol is removed. (A slight brown residue remains in the column but does not contain pregnanediol.) The extract is evaporated to dryness and transferred by means of a medicine dropper to a test tube with 95% alcohol. This is evaporated to a measured volume of 1-4 cc., depending upon the amount of pregnanediol expected, and exactly 3 volumes of water are added slowly while
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heating. This solution is boiled gently and allowed to cool slowly. When dealing with small quantities of pregnanediol, as in menstrual cycles, the solution is allowed to stand at room temperature for 24 hours in order to obtain maximum precipitation. The precipitate is collected on a sintered glass funnel with gentle suction. If the precipitate is not white, it is redissolved in 95% alcohol and reprecipitated and refiltered. When pure, the precipitate is dissolved in alcohol, transferred to a small previously weighed bottle placed in the oven to dry, and then weighed. PREGNANEDIOL EXCRETION IN THE MENSTRUAL CYCLE
Pregnanediol has been shown to be excreted during the menstrual cycle following ovulation and until 1-3 days prior to the onset of the menses. 5 , 9, 20, 32, 38, 43 For a time it was felt that the endometrium was necessary for the reduction of progesterone to pregnanediol. The evidence for this was the inability to isolate N aPG from the urine after the administration of progesterone in a woman who had had a hysterectomy,39 and the observation by Hamblen, Ashley, and Baptist that curettage during the luteal phase of the menstrual cycle was followed by an abrupt fall in N aPG excretion. However, Buxton and Westphal later recovered pregnanediol from the urine of men receiving progesterone and Venning and Browne,40 using larger doses of progesterone, succeeded in recovering pregnanediol from the urine of hysterectomized women and concluded that endometrium was not necessary for the conversion of progesterone to pregnanediol. OVULATION
U sing the method described, pregnanediol appears to be excreted within 24 hours after ovulation and continues to be excreted till the onset of menstruation. Figures 1 and 2 show pregnanediol excretion by normal subjects. In our experience28 pregnanediol excretion correlates poorly with temperature elevation, pregnanediol having appeared in the urine before, during, and after the temperature rise. In clinical usage daily collections of 24-hour urines are impractical so that it is the usual practice to rely on one or two measurements of urinary pregnanediol as an indication of ovulation. For this purpose specimens are best collected on Days 20 to 24 of the usual 28-30 day cycle. The advantages of pregnanediol excretion as a test of ovulation are its relative convenience, inexpensiveness to the patient, and the elimination of inadvertent interruption by endometrial biopsy of a much wanted preg-
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nancy. Studies of endometrial biopsy material when correlated with pregnanediol excretion indicate a close correlation, so that the finding of pregnanediol in the urine may be taken as evidence of secretory endometrium and active corpus luteum. The studies by Brown and Bradbury in 98jiiiif--------------~----_.~~
A.G. Fig. 1. Pregnanediol excretion in nonnal menstrual cycle. (Reprinted from Rogers, J., and Sturgis, S. H., J. Glin. Endocrinol. 10:89, 1950, by permission of Charles C Thomas, publisher.)
A.G. Fig. 2. Pregnanediol recovery in normal menstrual cycle. (Reprinted from Rogers, J., and Sturgis, S. H., /. Glin. Endocrinol. 10:89, 1950, by pennission of Charles C Thomas, publisher.)
prolonging corpus luteum activity by the use of chorionic gonadotrophin further indicate the correlation between corpus luteum activity, secretory endometrium, and pregnanediol excretion. Because of the necessity of serial collections of urine, pregnanediol excretion does not lend itself to the clinical study of the exact timing of ovulation except perhaps on a research basis.
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The problem of inadequate corpus lute urn function has been mentioned by several authors. Whether such an entity actually exists remains unsolved. There are some women (Fig. 3) whose pregnanediol excretion is lower than that normally encountered, suggesting the possibility that the corpus luteum of that particular cycle is deficient in the production of progesterone. 98.5
________________ ......,~BllIT~=r_._fi.iT~_ 3
Fig. 3. Diminished pregnanediol excretion. (Reprinted from Rogers, J., and Sturgis, S. H., ]. Glin. Endocrinol. 10:89, 1950, by permission of Charles C Thomas, publisher. )
PREGNANEDIOL EXCRETION IN PREGNANCY
The demonstration by Venningao . 36 of the pattern of excretion of sodium pregnanediol glucuronidate in pregnancy (Fig. 4) led to further observation by other workers and the hope was raised that pregnanediol excretion could be used as a reliable chemical test for pregnancy. However, a major drawback to its use as a pregnancy test is that the amount of pregnanediol recovered during the early weeks of pregnancy may not be sufficiently increased over that found in the luteal phase to be diagnostic. 43 However, good results have been reported in the measurement of pregnanediol excretion as a diagnostic aid in pregnancy.15-18 In a series of 248 cases Guterman reported 92 per cent accuracy in diagnosing pregnancy.16 THREATENED ABORTION
Although the measurement of pregnanediol has not been widely accepted as a pregnancy test, it has proved of considerable value in the prognosis of threatened abortion. Cope 8 was one of the first to point out that a falling
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level of pregnanediol in the urine might be a reliable indication of intrauterine death. Hamblen19 later reported on falling titers prior to abortion, and Guterman17 was able to prophesy the prognosis of threatened abortion in 93 per cent of 73 cases on the basis of a falling titer of pregnanediol when it should have been rising. "",.
so 40 50
- DAtS -
Fig. 4. (Courtesy E. H. Venning.) Excretion of various hormone metabolites in normal pregnancy, From Venning, E. H., Obst. & Gynec. Survey 8:661, 1948. (Reprinted courtesy of the author and the Williams & Wilkins Company, publishers.)
An interesting chapter in the development of our concepts of pregnanediol excretion started when the Smiths reported that diethylstilbestrol administered to pregnant women resulted in an increased excretion of pregnanediol as measured by the Venning method, and concluded that the estrogen administered stimulated progesterone secretion. This observation
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led to the popular use of stilbestrol in threatened abortion, habitual abortion, and pregnant diabetics. Subsequently, Davis and Fugo,ll using a method measuring pregnanediol in the free state, failed to confirm the observation of the Smiths that stilbestrol increased pregnanediol excretion. They pointed out that in the Venning method all the glucuronides are included in the final determination and suggested that diethylstilbestrol glucuronide as well as pregnanediol glucuronide had been included in the determinations reported by the Smiths. Sommerville and co-workers 30 came to similar conclusions and further demonstrated that in pregnancy the administration of diethylstilbestrol appears to decrease the excretion of pregnanediOl. Such observations have led to doubt concerning the value of stilbestrol during pregnancyP Measurement of pregnanediol excretion has been reported to be helpful in conditions associated with high gonadotrophic excretion other than pregnancy, such as moles and chorionepitheliomas, where pregnanediol may not be found in the urine, therefore serving to differentiate these conditions from pregnancyP However, de Watteville reported 3 cases of hydatid mole in which pregnanediol was present, rendering this differential point less secure. METABOLISM AND THEORETIC CONSIDERATIONS
The liver appears to be the site of conversion of progesterone to pregnanediol. This has been indicated by experiments in which progesterone pellets implanted into the spleen with rapid absorption through the liver were less effective than similar pellets placed in subcutaneous tissue. Masson and Hoffman demonstrated in rabbits that administration by gavage required 200 mg. of progesterone to obtain a progestational effect comparable to that elicited by 0.25 to 0.5 mg. administered subcutaneously. In the rabbit who had had a partial hepatectomy, however, comparable progestational changes were obtained by the administration of 25 mg. of progesterone by gavage. Pearlman and Pincus administered massive oral doses of ~5-pregnen-3,8 ol-20-one to a postmenopausal woman and recovered minute amounts of pregnane-3( a ),20( a )-diol, from the bile. Pearlman and Cerceo isolated pregnan-3( a )-ol-20-one and pregnane-3( a), 20(,8 )-diol from the gallbladder bile of pregnant cows. It has also been shown that after the oral administration of progesterone small amounts of pregnanediol could be
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isolated from the bile of human subjects in whom T-tube biliary drainage was established for the relief of common duct obstruction. 27 Such observations serve again to implicate the liver as the site of conversion of progesterone to pregnanediol. Measurements of pregnanediol excretion after a large oral dose of progesterone have been made in patients with various forms of parenchymal liver disease and in extrahepatic biliary obstruction. 26 The results are difficult to interpret. In some patients with hepatic disease pregnanediol excretion is markedly diminished, in a few it is increased, and in others it is normal. The results suggest that in some individuals with hepatic disease the metabolism of pregnanediol itself is interfered with but that in others the metabolic difficulty is in the conversion of progesterone to pregnanediol. In patients with obstructive disorders of the biliary tract significant increases in urinary pregnanediol may occur, suggesting that in biliary obstruction and interference with this pathway of excretion more pregnanediol is excreted correspondingly in the urine. SUMMARY AND CONCLUSIONS 1. Pregnanediol, the urinary metabolite of progesterone, can be measured by relatively simple methods. 2. The finding of pregnanediol in the urine of women in the luteal phase can be accepted as evidence of ovulation. 3. The measurement of pregnanediol as a pregnancy test is of value in some instances, but is helpful chiefly in aiding in the prognosis of threatened abortion. 4. Some aspects of the relation of progesterone metabolism to the liver have been briefly discussed. REFERENCES 1. 2. 3. 4. 5.
ASTWOOD, E. B., GRADY, A. B., and WERMER, O. S. Cited by Rogers, J., and Sturgis, S. H.28 ASTWOOD, E. B., and JONES, G. E. S. A simple method for the quantitative determination of pregnanediol in human urine. J. Bioi. ehem. 137:397, 1941. BROWN, W. E., and BRADBURY, J. T. A study of the physiologic action of human chorionic hormone. Am.]. Obst. & Gynee. 53:749, 1947. BUTENANDT, A. Dber das pregnandiol, einen neuen Sterin-abkommling aus Schwangeren-Harn. Ber. ehem. Gesellseh. 63:659, 1930. BUXTON, C. L. Pregnanediol determination as an aid in clinical diagnosis. Am. ]. Obst. & Gynee. 40:202, 1940.
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6. BUXTON, C. L., and WESTPHAL, U. Recovery of pregnanediol in urine of men treated with progesterone. Proc. Soc. Exper. Biol. & Med. 41 :284, 1939. 7. CHANEY, A. L., MCKEE, M. S., FISCHER, R. H., and MCCoLGAN, S. P. A new and simplified procedure for the determination of free pregnanediol in urine and its evaluation. ]. Clin. Endocrinol. & Metab. 12:735, 1952. 8. COPE, C. L. The diagnostic value of pregnanediol excretion in pregnancy disorders. Brit. M. ]. 2:545, 1940. 9. COPE, C. L. The excretion of pregnanediol and the corpus luteum. Clin. Sc. 4:217, 1940. 10. DAVIS, M. E., and FUGo, N. W. A simplified method for the quantitative determination of free pregnanediol excretion in pregnancy. Proc. Soc. Exper. Biol. & Med. 66:39, 1947. II. DAVIS, M. E., and FUGo, N. W. Does administration of diethylstilbestrol to pregnant women result in increased output of urinary pregnanediol? Proc. Soc. Exper. . Biol. & Med. 69:436, 1948. 12. DIECKMANN, W. J., DAVIS, M. E., RYNKIEWICZ, S. M., and POTTINGER, R. E. Does the administration of diethylstilbestrol during pregnancy have therapeutic value? Am. ]. Obst. & Gynec. 66:1062, 1953. 13. DOSNE, C. Inactivation of antifibromatogenic substances (progesterone and desoxycorticosterone acetate) in the liver. Cancer Research 4:512, 1944. 14. GOLDFINE, M. M., and COHEN, S. L. Glucuronidase hydrolysis for pregnanediol assays. Endocrinology 52:597, 1953. 15. GUTERMAN, H. S. A human pregnancy test based upon a color reaction of pregnanediol in the urine. J. Clin. Endocrinol. 4:262, 1944. 16. GUTERMAN, H. S. Further observations on the value of the pregnanediol test for pregnancy. ]. Clin. Endocrinol. 5:407, 1945. 17. GUTERMAN, H. S. Prediction of fate of threatened abortion by pregnanediol. ].A.M.A. 181 :378, 1946. 18. GUTERMAN, H. S., and SCHROEDER, M. S. A simplified technique for the quantitative colorimetric estimation of pregnanediol in urine. J. Lab. & Clin. Med. 88: 356, 1948. 19. HAMBLEN, E. C. Some clinical observations on the endocrinology of abortion. Am. ]. Obst. & Gynec. 41 :664, 1941. 20. HAMBLEN, E. C., ASHLEY, C., and BAPTIST, M. Sodium pregnanediol glucuronide: The significance of its excretion in the urine. Endocrinology 24: 1, 1939. 2l. HUBER, D. Determination of pregnanediol in urine for diagnostic purposes. Biochem. ]. 41 :609, 1947. 22. MARRIAN, G. F. Chemistry of oestrin: Preparation from urine and separation from unidentified solid alcohol. Biochem.]. 28:1090, 1929. 23. MASSON, G., and HOFFMAN, M. M. Studies on the role of the liver in the metabolism of progesterone. Endocrinology 87: Ill, 1945. 24. PEARLMAN, W. H., and CERCEO, E. The isolation of pregnanol-3 (a) -one-20, pregnanediol-3(a),20(b) and etiocholanediol-3(a),17(b) from the bile of pregnant cows. ]. BioI. Chem. 176:847, 1948. 25. PEARLMAN, W. H., and PINCUS, G. The metabolism of pregnenolone. Fed. Proc. 5:79,1946. 26. ROGERS, J. Unpublished data. 27. ROGERS, J., and McLELLAN, F. Isolation of pregnanediol from human bile after oral administration of progesterone. J. Clin. Endocrinol. 11 :246, 1951. 28. ROGERS, J., and STURGIS, S. H. Pregnanediol excretion in normal women. ]. Clin. Endocrinol. lO:89, 1950.
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29. SMITH, O. W., SMITH, G. V., and HURWITZ, D. Increased excretion of pregnanediol in pregnancy from diethylstilbestrol, with special reference to prevention of late pregnancy accidents. Am.]. Gbat. & Cynec. 51 :411, 1946. 30. SOMMERVILLE, 1. F., MARRIAN, G. F., and CLAYTON, B. E. Effect of diethylstilboestrol on urinary excretion of pregnanediol and endogenous oestrogen during pregnancy. Lancet 256:680, 1949. 31. SOMMERVILLE, 1. F., MARRIAN, G. F., and KELLAR, R. J. Rapid determination of urinary pregnanediol. Lancet 2:89, 1948. 32. STOVER, R. F., and PRATT, J. P. Progestin studies: Pregnanediol excretion. Endocrinology 24:29, 1939. 33. TALBOT, N. B., BERMAN, R. A., McLACHLAN, E. A., and WOLFE, J. K. The colorimetric determination of neutral steroids (hormones) in a 24 hour sample of human urine (pregnanediol; total alpha and beta alcoholic and non-alcoholic 17-ketosteroids). /. Clin. Endocrinol.1:668, 1941. 34. VENNING, E. H. Gravimetric method for the determination of sodium pregnanediol glucuronidate (an excretion product of progesterone). /. BioI. Chern. 119:473, 1937. 35. VENNING, E. H. Further studies on estimation of small amounts of sodium pregnanediol glucuronidate in urine. /. Bioi. Chern. 126:595, 1938. 36. VENNING, E. H. Excretion of various hormone metabolites in normal pregnancy. Gbat. & Cynec. Survey 8:661, 1948. 37. VENNING, E. M., and BROWNE, J. S. L. Isolation of a water soluble pregnanediol complex from human pregnancy urine. Proc. Soc. Exper. Bioi. & Med. 84:792, 1936. 38. VENNING, E. H., and BROWNE, J. S. L. The urinary excretion of sodium pregnanediol glucuronidate in the human menstrual cycle. EndOCrinology 21 :711, 1937. 39. VENNING, E. H., and BROWNE, J. S. L. A study of the metabolism of progesterone. Am./. Physiol. 128:209, 1938. 40. VENNING, E. H., and BROWNE, J. S. L. A study of the metabolism of crystalline progesterone. EndOcrinology 27:707, 1940. 41. VENNING, E. M., HENRY, J. S., and BROWNE, J. S. L. The measurement of a pregnanediol complex in human urine. Canad. M. A. ]. 86:83, 1937. 42. DE WATTEVILLE, H. Pregnanediol determinations in the clinic and in research. /. Clin. Endocrinol. 11 :251, 1951. 43. WILSON, R. B., RANDALL, L. M., and OSTERBERG, A. E. Studies on pregnanediol. Am./. Gbat. & Cynec. 87:59, 1939.